Journal of microbiological methods最新文献

{"title":"Comparative methods to identify variations in membrane fatty acids reflecting microbial community differences","authors":"Petra Lovecká ,&nbsp;Blanka Vrchotová ,&nbsp;Anna Košinová ,&nbsp;Jan Riegert","doi":"10.1016/j.mimet.2025.107283","DOIUrl":"10.1016/j.mimet.2025.107283","url":null,"abstract":"<div><div>This study presents a novel application of fatty acid methyl ester (FAME) profiling to monitor and characterize seasonal and spatial dynamics in the endophytic microbial communities of grapevine (<em>Vitis vinifera</em>). Using gas chromatography with flame ionization detection, we quantified selected FAMEs in leaves and stems from four grapevine varieties sampled across different seasons and farming systems (biodynamic vs. conventional). Our results show that plant organ type and season are the dominant factors shaping endophytic community composition and abundance, with spring leaves harbouring the highest levels and winter stems the lowest. This approach provides a rapid, cultivation-independent method for detecting shifts in endophytic communities, offering new opportunities for monitoring plant health, evaluating sustainable viticulture practices, and identifying conditions that foster beneficial plant–microbe interactions.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107283"},"PeriodicalIF":1.9,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transcriptomic insights into biofilm dynamics and therapeutic targets in chronic wound infections (MIMET 107281)","authors":"Omprakash Pendor,&nbsp;Sejal Ukey,&nbsp;Rashmi Trivedi,&nbsp;Milind Umekar","doi":"10.1016/j.mimet.2025.107281","DOIUrl":"10.1016/j.mimet.2025.107281","url":null,"abstract":"<div><div>Chronic wound infections remain a significant clinical challenge, primarily due to the formation of bacterial biofilms that hinder healing and increase antimicrobial resistance. This review explores various studies focused on biofilm development, transcriptional responses, and therapeutic strategies for combating biofilm-associated infections. Using <em>Pseudomonas aeruginosa</em> PAO1 in ex vivo porcine skin wound models, RNA-seq analysis revealed key genes involved in biofilm formation, notably the <em>lapA</em> gene encoding alkaline phosphatase, which was upregulated, while denitrification pathway genes such as <em>nirS</em> were downregulated. Targeting these pathways through NO induction showed potential for biofilm disruption.</div><div>Novel biofilm-inhibiting strategies, including silver nanoparticles, lactoferrin, and exopolysaccharides, demonstrated antibacterial, anti-biofilm, and wound-healing effects. Additionally, metal-based nanozymes (Ru-procyanidin nanoparticles) and microneedle patches embedded with cerium/zinc composites emerged as promising solutions for oxidative stress reduction and bacterial elimination in diabetic wounds. Omics approaches, particularly transcriptomics and metabolomics, have further elucidated biofilm differentiation mechanisms and host-pathogen interactions.</div><div>Advanced detection methods such as electrochemical biosensors and peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) have improved the identification of biofilm-associated infections. Furthermore, comparative analyses of mixed-species and single-species biofilms highlighted their differential impact on wound healing, with polybacterial biofilms causing more severe impairment.</div><div>These findings underscore the importance of integrating RNA-based diagnostics, molecular therapies, and novel biomaterials to enhance chronic wound management. Future research should focus on translating these insights into clinical applications for more effective biofilm-targeted treatments.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107281"},"PeriodicalIF":1.9,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206642","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Performance qualification of impedance flow cytometry as a rapid in-process control proxy for Colony-forming units in bacterial fermentation processes","authors":"Peter Lüttge Jordal,&nbsp;Marcos González Díaz,&nbsp;Frederik Aalund,&nbsp;Gustav Skands","doi":"10.1016/j.mimet.2025.107284","DOIUrl":"10.1016/j.mimet.2025.107284","url":null,"abstract":"<div><div>Impedance flow cytometry (IFC) was evaluated against colony-forming units (CFU) for six bacterial genera. Across exponential, deceleration, and stationary phases, IFC correlated near-perfectly with CFU method (R² ≈ 1). Divergence occurred in death stage. IFC supports decision-making on when to transfer and harvest bacteria in active fermentation processes.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107284"},"PeriodicalIF":1.9,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145206655","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hyphal induction in Candida albicans: Optimizing medium parameters to accelerate hyphal growth enhanced by GlcNAc","authors":"Rachana A. ,&nbsp;Atheena P.V. ,&nbsp;Allan J. Britto ,&nbsp;Uttara Chakraborty ,&nbsp;Ritu Raval","doi":"10.1016/j.mimet.2025.107282","DOIUrl":"10.1016/j.mimet.2025.107282","url":null,"abstract":"<div><div>Hyphal formation is a key virulence trait in <em>Candida albicans,</em> playing a crucial role in the development and progression of candidiasis. As a result, it remains a significant area of interest for antifungal drug target studies. Conventional hyphal induction medium typically contain multiple components, making them complex to prepare. In this study, we have optimized a simple and cost-effective medium with minimal components that induces hyphal formation. Among different combination, MF8 with Peptone 0.16 %, Dextrose 0.4 % and BSA 0.25 % caused moderate hyphal formation. Further addition of <em>N</em>-acetylglucosamine (GlcNAc) enhanced hyphal formation and caused clumped mycelial growth. To determine the different combinations of GlcNAc and MgSO₄ that cause hyphal formation, we employed a Design of Experiment (DOE) approach using JMP software. GlcNAc was found to be a significant component in hyphal induction (<em>p</em> &lt; 0.05, R² = 0.26) and MgSO₄ was not significant (<em>p</em> &gt; 0.05). Further gene expression and pH was analysed under Run 20 conditions (30 mM GlcNAc, 1 mM MgSO₄) which revealed significant transcriptional activation of hyphal-specific genes hyphal wall protein 1(<em>HWP1</em>) and Hypha-specific G1 cyclin 1(<em>HGC1</em>) in the optimized medium. Notably, <em>HWP1</em> expression increased 100-fold at 4 h and 85-fold at 6 h (<em>p</em> &lt; 0.05), while <em>HGC1</em> showed a 7.09-fold and 6.07-fold upregulation at the same time points (<em>p</em> &lt; 0.05). Additionally, there was an initial shift toward alkalinity at 1 and 2 h time point and a drop to acidic pH at 4 h and 6 h time point suggesting hyphal formation and upregulation of hyphal specific genes induced with the lowering of pH in medium.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107282"},"PeriodicalIF":1.9,"publicationDate":"2025-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145199730","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A multiplexed TSA/CRISPR-mediated one-pot system for rapid detection of high-risk animal-derived infectious diseases","authors":"Zhenghua Gong ,&nbsp;Tian Lu ,&nbsp;Zhouxi Ruan ,&nbsp;Rongrong Zhang ,&nbsp;Songqi Zhu ,&nbsp;Zhenyao Xia ,&nbsp;Jianfeng Zhong ,&nbsp;Guilan Wang ,&nbsp;Yingxin Li ,&nbsp;Qian He ,&nbsp;Rongqi Liu ,&nbsp;Jun Che","doi":"10.1016/j.mimet.2025.107277","DOIUrl":"10.1016/j.mimet.2025.107277","url":null,"abstract":"<div><div>The importance of rapid and convenient pathogen detection has been emphasized by the alarming threat of the Coronavirus Disease 2019 (COVID-19) pandemic since 2019. Point-of-care testing (POCT) provides rapid diagnostic results directly at the sampling site. However, isothermal amplification-based POCT faces technical challenges including primer design complexity and false-positive rates. To address these limitations, we developed the Thermostatic Step Amplification (TSA)/Clustered regularly interspaced short palindromic repeats (CRISPR) One-Pot System (TCOPS). This sensitive, rapid, and efficient platform specifically detects Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Mpox virus (MPXV) and Rabíes virus (RV) through integrated amplification and CRISPR-based detection. Our integrated TCOPS overcomes the technical challenges through single-tube reactions combining thermostatic amplification and CRISPR detection, reducing contamination while maintaining high accuracy for field applications. TCOPS enables single-tube CRISPR detection of high-risk viruses, with 10 copies/μL sensitivity shown using cloned DNA template for RV. In evaluations against Quantitative Polymerase Chain Reaction (qPCR) using 50 clinical samples, TCOPS incorporating freeze-dried reagents and a newly developed miniature fluorescence system (Q max) demonstrated &gt;90 % sensitivity and 100 % specificity. Combined with the portable Q max device and its lyophilized reagent kit, TCOPS enables simple, rapid detection of multiple zoonotic viruses (SARS-CoV-2, MPXV, and RV) at the point of care. This integrated system achieves high sensitivity and specificity while establishing a practical, field-deployable prototype for next-generation POCT applications in resource-limited settings.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107277"},"PeriodicalIF":1.9,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145156103","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of Manuka Honey microbubble particle structures for enhanced inhibition of Pseudomonas aeruginosa growth","authors":"Pei-Ju Lin","doi":"10.1016/j.mimet.2025.107278","DOIUrl":"10.1016/j.mimet.2025.107278","url":null,"abstract":"<div><div>This study aims to optimize the use of Manuka Honey (MH) microbubbles for enhancing antimicrobial efficacy in the treatment of <em>Pseudomonas aeruginosa</em> (<em>P. aeruginosa.</em>) infections. The experimental design included three different MH microbubble particle sizes produced by three types of high-density stainless steel mesh layers, two types of dressing, two volumes of use, and three antimicrobial exposure times. The bacterial survival percentages of <em>P. aeruginosa</em> survival obtained under each combination of variables was calculated. The results demonstrated that MH microbubble size and antimicrobial exposure time were the key factors that improved growth inhibition. Additionally, the use of a high-density stainless steel mesh nozzle to produce MH microbubbles significantly enhanced inhibition of bacterial growth, with smaller microbubble sizes showing better inhibition (<em>p</em> &lt; 0.05). Complete bacterial elimination (3.5 log) was achieved using either waterproof or foam dressings with three of nozzle layers (producing the microbubbles of 0.0016 mm) and 3 mL of microbubbles over 24 h.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107278"},"PeriodicalIF":1.9,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145175645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advancement in microalgal metabolites extraction techniques: Multi-factorial considerations","authors":"Arya Mishra ,&nbsp;Mamta Bhandari ,&nbsp;Tarun Pant ,&nbsp;Harshit Tiwari ,&nbsp;Ajay Kumar Kataria ,&nbsp;Vijay Khandelwal ,&nbsp;Naveen Chand ,&nbsp;Sanjeev Kumar Prajapati","doi":"10.1016/j.mimet.2025.107280","DOIUrl":"10.1016/j.mimet.2025.107280","url":null,"abstract":"<div><div>Microalgae are a rich source of high-value metabolites such as lipids, pigments, proteins, and polysaccharides, yet their recovery is limited by the complexity of cell wall structures and costly downstream processing. This review evaluates advanced green extraction techniques including enzyme-assisted (EAE), ionic liquid (ILs), microwave-assisted (MAE), milking, pressurized liquid (PLE), pulsed electric field (PEF), supercritical fluid (SFE), and ultrasound-assisted extraction (UAE). Here we also provide a multi-factorial comparative analysis incorporating cost, scalability, extraction efficiency, and environmental impact. The results indicate that EAE and SFE achieve the highest yields (up to 97 %) but are constrained by cost, whereas UAE and PEF balance scalability and sustainability, with UAE reducing extraction time by up to 60 % compared to conventional methods. Integrated approaches (e.g., ILs + SFE, UAE + EAE) show promising synergies, enhancing yield while reducing solvent demand. Overall, this review offers research advancements and insights on selecting the most appropriate extraction strategy depending on industrial priorities, thereby advancing sustainable commercialization of microalgal metabolites.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107280"},"PeriodicalIF":1.9,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145176150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of culture medium for probiotic bacterium Lacticaseibacillus casei UT1 to enhance biomass production using the Taguchi statistical method","authors":"Saman Afshar ,&nbsp;Gholamreza Zarrini ,&nbsp;Nader Farsad-Akhtar","doi":"10.1016/j.mimet.2025.107279","DOIUrl":"10.1016/j.mimet.2025.107279","url":null,"abstract":"<div><div>Probiotics offer many benefits and have attracted significant research interest in recent years. Given their growing applications, large-scale mass production is essential for various industries. However, conventional media for <em>Lactobacilli</em> cultivation, such as MRS (de Man, Rogosa, Sharpe) and skim milk, contain expensive compounds and are therefore not ideal for probiotic-based industries. Consequently, developing a medium with low-cost ingredients, such as molasses and whey, is of utmost importance. To address this need, various carbon and nitrogen sources were first investigated using a completely randomized factorial design (CRFD) to optimize an industrial culture medium for enhancing biomass production of <em>Lacticaseibacillus casei</em> UT1. This screening revealed molasses + whey (MW) as the optimal carbon source and yeast extract (YE) as the optimal nitrogen source. Building on these results, the Taguchi method was then applied to evaluate these two components alongside five additional factors. The experimental outcomes were analyzed using statistical methods to determine the optimal conditions. According to the Taguchi design, the optimum formulation for biomass production of L. <em>casei</em> UT1 was found to be 8 % (<em>w</em>/<em>v</em>) MW, 4 % (w/v) YE, 0.04 % (w/v) magnesium sulfate, and 0.005 % (w/v) manganese sulfate. Under these optimized conditions, biomass production increased by approximately 112 % compared to the MRS medium, as predicted by the Taguchi method and confirmed experimentally.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107279"},"PeriodicalIF":1.9,"publicationDate":"2025-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145175541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In silico identification and field validation of diagnostic marker gene targets for the improved detection of scrub typhus","authors":"Akash Balasaheb Mote ,&nbsp;Himani Dhanze ,&nbsp;Prasad Thomas ,&nbsp;M. Suman Kumar ,&nbsp;Vibha Singh ,&nbsp;Azhahianambi Palavesam ,&nbsp;Rajeev Singh ,&nbsp;Sandeep P. Chaudhari ,&nbsp;K.P. Singh ,&nbsp;Hira Ram ,&nbsp;Nagendra R. Hegde ,&nbsp;G. Taru Sharma","doi":"10.1016/j.mimet.2025.107274","DOIUrl":"10.1016/j.mimet.2025.107274","url":null,"abstract":"<div><div>Scrub typhus, a vector-borne zoonosis prevalent in the Asia-Pacific region, poses diagnostic challenges due to the pathogen's complex genome and diverse rodent and shrew hosts. The scarcity of reliable diagnostic tests hinders effective sentinel surveillance. This study aims to identify novel diagnostic gene targets using a bioinformatics approach to develop a highly sensitive and specific PCR assay for scrub typhus detection. Genome sequences of <em>Orientia tsutsugamushi</em>, the causative agent, were analyzed, leading to the selection of 11 potential diagnostic biomarkers. In-house conventional PCR assays targeting these biomarkers and published nested PCR assays, were tested on blood and tissue (spleen) samples of 150 field rodent and shrew. Among the tested genes, the <em>tsa56</em> gene consistently demonstrated the highest detection rate in both conventional (55.6 %, <em>n</em> = 15) and nested (74 %, <em>n</em> = 20) assays, indicating it to be the most reliable diagnostic marker for scrub typhus. A novel nested PCR was designed targeting a unique <em>tsa56</em> gene segment, which showed an analytical sensitivity of 4.7 (95 % CI: 2–7.4) copies/μL, and was able to detect <em>O. tsutsugamushi</em> in multiple hosts including human and mite samples. Moreover, fewer primer-template mismatches and no mismatches at the critical 3′ terminus with <em>O. tsutsugamushi</em> strains were observed. The assay did not show any cross-reaction with available non-target organisms. Thus, the developed nested PCR assay demonstrates enhanced sensitivity, specificity, and broader strain inclusivity. Overall, the study presents a promising tool for scrub typhus detection, which will aid in improved disease surveillance, outbreak prediction, and timely implementation of control measures.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107274"},"PeriodicalIF":1.9,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanobubble water for the effective removal of biofilm formed by Escherichia coli","authors":"Fumiyuki Kobayashi ,&nbsp;Takeru Kawahara ,&nbsp;Asako Narai-Kanayama ,&nbsp;Sachiko Odake","doi":"10.1016/j.mimet.2025.107276","DOIUrl":"10.1016/j.mimet.2025.107276","url":null,"abstract":"<div><div>The removal of biofilm (BF) formed by <em>Escherichia coli</em> using nanobubble (NB) water prepared with different gases and two generation methods was investigated. The <em>E. coli</em> BF removal efficiencies of all NB water samples tested were significantly higher than those of the non-NB water. In particular, nitrogen (N₂) NB water exerted the greatest effect on the removal of <em>E. coli</em> BF. The surface tension of N₂NB water was the lowest, although that of airNB water was the highest. The dissolved oxygen (O₂) concentration in the O₂NB water was drastically increased, although that in the N₂NB and carbon dioxide (CO₂) NB water was decreased. The pH of CO₂NB water was significantly decreased, although that of N₂NB, airNB, and O₂NB water was increased. The <em>E coli</em> BF removal efficiencies of N₂NB water were not different in the NB generators between ejector and shearing types. However, the surface tension, pH, and size distribution varied among the NB generator types. Therefore, it was found that NB water removed <em>E. coli</em> BF, and that its efficiencies differed depending on the gas type.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"238 ","pages":"Article 107276"},"PeriodicalIF":1.9,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145137889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}